This work was supported by the grants from the National Natural Science Foundation of China (31401013 and 31471010), the Science and Technology Commission of Shanghai Municipality, Shanghai Pujiang Program (14PJ1405900), and Natural Science Foundation of Shanghai (19ZR1446400).

1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster16
<ol>
	<li>Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.</li>
	<li>Prepare 500 &#181;L of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample.</li>
	<li>Anesthetize adult flies with CO2 on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for example, fix the fly thoraxes by a pair of forceps, and then isolate the fly abdomens by cutting along the border of the thorax and abdomen using a pair of scissors. Collect the fly thoraxes for the muscle citrate synthase activity assay. 
	NOTE: Determine the fresh weight of tissue at this step if using it to normalize citrate synthase activity.</li>
	<li>Transfer 10 adult fly thoraxes to 100 &#181;L of ice-cold extraction buffer immediately and homogenize with a pestle on ice. To keep the samples ice-cold, homogenize the samples for 5-10 s on ice, then have the samples sit on ice for 5 s, and repeat until all the tissues in the tube are homogenized completely. 
	NOTE: Tape the tube, check the homogenates to make sure that there are no clots in the homogenates and that the tissues in the tube are homogenized completely. All sample treatments should be performed on ice.</li>
	<li>Take 10 &#181;L of each homogenized sample in a new tube for protein content measurement. Keep the samples for protein measurement on ice. 
	NOTE: The protein samples can be frozen and stored at -80 &#176;C for later analysis. The protein concentrations serve as an internal parameter for normalization of citrate synthase activities.</li>
	<li>Add 400 &#181;L of ice-cold extraction buffer to each remaining sample to make a total volume of 500 &#181;L. Mix well by gently pipetting up and down at least 5x, avoiding bubble formation. For each reaction, add 1 &#181;L of diluted cell lysate to 150 &#181;L of the freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl CoA, 1 mM oxaloacetic acid). Mix thoroughly and immediately by gently pipetting, avoiding bubble formation. Measure the absorbance at 412 nm every 10-30 s for 4 min at 25 &#176;C using a plate reader capable of measuring absorbance at 412 nm at minimum intervals of 10 s. 
	NOTE: Change the tip before pipetting a different reagent and avoid forming bubbles in the wells. Incomplete mixing of the reagents may cause variations in the measurements. The citrate synthase activity assay should be done at room temperature immediately after the samples are collected, as this assay is used to quantify intact mitochondrial mass. The reaction buffer should be prepared immediately before use and should not be stored.</li>
	<li>Plot data as optical density (OD) absorbance (abs) (Y-axis) versus time (in minutes) (X-axis). Then calculate the slope for the linear portion of the curve, where the reaction rate is 
	 
	<img src="/files/ftp_upload/59454/59454eq1.jpg" /> 
	 
	Divide the value by the sample protein concentration to normalize the citrate synthase activity. The citrate synthase activity is calculated as 
	 
	<img src="/files/ftp_upload/59454/59454eq2.jpg" /> 
	 
	NOTE: The sample protein concentration can be measured by a protein concentration assay kit.</li>
</ol>

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The authors declare no conflicts of interest.

Metabolic studies using Drosophila as a model must take into consideration the genetic background, diet, and stock maintenance of the flies18. To avoid the effects of different genetic backgrounds on the measurement of citrate synthase activity, different strains of Drosophila should be backcrossed to the control strain for 10 generations. The genetic background of all Drosophila strains used in our experiments is w1118, so we used w1118</sup...

Mitochondria are best known as the power-producing organelles in most eukaryotic organisms, which produce the energy currency, ATP, through the tricarboxylic acid cycle (i.e., Krebs cycle) and oxidative phosphorylation. Mitochondria are also found to play important roles in a lot of other physiological processes, such as regulation of apoptosis1, Ca2+ homeostasis2,3, reactive oxidation species (ROS) generation4, and endoplasmic reticulum (ER)-stress response5. Mitochondrial dysfunction can affect any organ in the body at any age and is a primary cause of metabolic, aging-related6, and neurodegenerative diseases7. Intact mitochondria are mechanistically related to mitochondrial function. Thus, proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function8. Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle9, is localized in the mitochondrial matrix within eukaryotic cells, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass9,10. Citrate synthase activity can also be used as a normalization factor for intact mitochondrial proteins11,12.
The fruit fly, Drosophila melanogaster, is an excellent model system for studying mitochondrial function, as many molecules and pathways that play pivotal roles in mitochondria are evolutionarily conserved from Drosophila to humans13,14,15. Here, we present a protocol for a fast and simple method for measurement of citrate synthase activity by a colorimetric assay in Drosophila tissue homogenates16 in a 96 well plate format. In the citrate synthase activity assay, citrate synthase in Drosophila tissue homogenate catalyzes the reaction of oxaloacetate with acetyl coenzyme A (acetyl CoA) to form the citrate CoA-SH and H+. CoA-SH subsequently reacts with 5,5&#39;-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate a colored product, 2-nitro-5-thiobenzoate (TNB), which can be easily measured spectrophotometrically at 412 nm. Citrate synthase activity can be reflected by the rate of color production.

<table><tbody><tr><td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>V900477</td><td></td></tr><tr><td>2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base)</td><td>Sigma-Aldrich</td><td>V900483</td><td></td></tr><tr><td>Acetyl-CoA</td><td>Sigma-Aldrich</td><td>A2181</td><td></td></tr><tr><td>Dithio-bis-nitrobenzoic acid (DTNB)</td><td>Sigma-Aldrich</td><td>D8130</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>V900106</td><td></td></tr><tr><td>Oxaloacetate</td><td>Sigma-Aldrich</td><td>O4126</td><td></td></tr><tr><td>Pellet pestle</td><td>Sangon</td><td>F619072</td><td></td></tr><tr><td>Pellet pestle motor</td><td>Tiangen</td><td>OSE-Y10</td><td></td></tr><tr><td>Plate reader</td><td>BioTek</td><td>Eon</td><td></td></tr><tr><td>Protein BCA Assay kit</td><td>Beyotime</td><td>P0010S</td><td></td></tr><tr><td>Scissors</td><td>WPI</td><td>14124</td><td></td></tr><tr><td>Triton X-100</td><td>Sangon</td><td>A110694-0100</td><td></td></tr></tbody></table>

a colorimetric assay of citrate synthase activity in drosophila melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

Figure 1 presents an example of the kinetic curves for the OD absorbance at 412 nm over time obtained using the citrate synthase activity colorimetric assay to measure the Drosophila thorax tissue homogenates of different genotypes. It is well known that PGC-1&#945; is a master regulator of mitochondrial biogenesis. PGC-1&#945; is functionally conserved between Drosophila and humans. Drosophila RNF34 (dRNF34) is an E3 ubiquitin liga...

Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

a-colorimetric-assay-citrate-synthase-activity-drosophila

Research

JoVE Journal

Biochemistry

10.0K Views.  Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Drosophila dient als goed modelsysteem voor het bestuderen van mitochondriale functie. Hier presenteren we een protocol voor het meten van citraatsyndering in weefselhomogenate van volwassen vliegen. Dit protocol vereist geen isolatie van mitochondriën.Het is snel en eenvoudig en het is geschikt voor het meten van citraatsyntheseactiviteit in larven, gekweekte cellen en weefsels van zoogdieren. Begin met het verzamelen van volwassen vliegen voor elk monster, ervoor te zorgen dat ten m...

Video: A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Neuroscience

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Biology

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Adapting Microrespirometry for Drosophila - Insights into CASK Mutant Metabolism

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

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Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

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